No abstract

Previously, we have isolated and characterized an enhancer from the 5'-flanking region of the adipocyte P2 (aP2) gene that directs high-level adipocyte-specific gene expression in both cultured cells and transgenic mice. The key regulator of this enhancer is a cell type-restricted nuclear factor termed ARF6. Target sequences for ARF6 in the aP2 enhancer exhibit homology to a direct repeat of hormone response elements (HREs) spaced by one nucleotide; this motif (DR-l) has been demonstrated previously to be the preferred binding site for heterodimers of the retinoid X receptor (RXR) and the peroxisome proliferator-activated receptor (PPAR). We have cloned a novel member of the peroxisome proliferator-activated receptor family designated mPPART2, and we demonstrate that a heterodimeric complex of mPPAR~/2 and RXR~ constitute a functional ARF6 complex. Expression of mPPAR,/2 is induced very early during the differentiation of several cultured adipocyte cell lines and is strikingly adipose-specific in vivo. mPPAR-/2 and RXRc~ form heterodimers on ARF6-binding sites in vitro, and antiserum to RXRc~ specifically inhibits ARF6 activity in adipocyte nuclear extracts. Moreover, forced expression of mPPAR~/2 and RXR~ activates the adipocyte-specific aP2 enhancer in cultured fibroblasts, and this activation is potentiated by peroxisome proliferators, fatty acids, and 9-cis retinoic acid. These results identify mPPAR~/2 as the first adipocyte-specific transcription factor and suggest mechanisms whereby fatty acids, peroxisome proliferators, 9-cis retinoic acid, and other lipids may regulate adipocyte gene expression and differentiation.

Adipose differentiation is accompanied by changes in cellular morphology, a dramatic accumulation of intracellular lipid and activation of a specific program of gene expression. Using an mRNA differential display technique, we have isolated a novel adipose cDNA, termed adipoQ. The adipoQ cDNA encodes a polypeptide of 247 amino acids with a secretory signal sequence at the amino terminus, a collagenous region (Gly-X-Y repeats), and a globular domain. The globular domain of adipoQ shares significant homology with subunits of complement factor C1q, collagen ␣1(X), and the brainspecific factor cerebellin. The expression of adipoQ is highly specific to adipose tissue in both mouse and rat. Expression of adipoQ is observed exclusively in mature fat cells as the stromal-vascular fraction of fat tissue does not contain adipoQ mRNA. In cultured 3T3-F442A and 3T3-L1 preadipocytes, hormone-induced differentiation dramatically increases the level of expression for adipoQ. Furthermore, the expression of adipoQ mRNA is significantly reduced in the adipose tissues from obese mice and humans. Whereas the biological function of this polypeptide is presently unknown, the tissue-specific expression of a putative secreted protein suggests that this factor may function as a novel signaling molecule for adipose tissue.Adipose tissue is highly specialized to play important roles in energy storage, fatty acid metabolism, and glucose homeostasis (1, 2). Adipocytes synthesize and store triglyceride in periods of nutritional abundance and mobilize the lipids in response to fasting (2, 3). Fat tissue is also involved in regulating blood glucose levels through the expression of the insulin responsive glucose transporter, Glu4 (4, 5). Fat and muscle, in fact, constitute the two major sites for insulin-regulated glucose uptake.At a molecular level, many genes involved in lipid metabolism and glucose homeostasis are prominently expressed in fat (1). These include fatty acid synthase (6), the fatty acid binding protein aP2 (7,8), lipoprotein lipase (9), phosphoenolpyruvate carboxykinase (10), malic enzyme (11), glyceraldehyde-3-phosphate dehydrogenase (12), and Glut4 (4). Receptors for lipogenic or lipolytic hormones such as insulin (13, 14), insulin-like growth factor 1 (15), and adrenergic compounds (16,17) are also expressed in adipocytes. In addition to these genes that clearly participate in the metabolic functions of adipose tissue, a group of genes that function in extracellular signaling have also been identified in fat. A prototype of these molecules is insulin-like growth factor 1, which is expressed in many tissues during development and plays an important role in cell proliferation (18). In adipocytes, however, insulin-like growth factor 1 is found to stimulate cell differentiation (19). More interestingly, insulin-like growth factor 1 is synthesized by preadipocytes in response to growth hormone stimulation (20), thus potentially functioning in an autocrine or paracrine fashion to promote adipogenesis during development. Another si...

Adipocyte differentiation is an important component of obesity and other metabolic diseases. This process is strongly inhibited by many mitogens and oncogenes. Several growth factors that inhibit fat cell differentiation caused mitogen-activated protein (MAP) kinase-mediated phosphorylation of the dominant adipogenic transcription factor peroxisome proliferator-activated receptor γ (PPARγ) and reduction of its transcriptional activity. Expression of PPARγ with a nonphosphorylatable mutation at this site (serine-112) yielded cells with increased sensitivity to ligand-induced adipogenesis and resistance to inhibition of differentiation by mitogens. These results indicate that covalent modification of PPARγ by serum and growth factors is a major regulator of the balance between cell growth and differentiation in the adipose cell lineage.

Skeletal muscle and adipose tissue development often has a reciprocal relationship in vivo, particularly in myodystrophic states. We have investigated whether determined myoblasts with no inherent adipogenic potential can be induced to transdifferentiate into mature adipocytes by the ectopic expression of two adipogenic transcription factors, PPARy and C/EBPa. When cultured under optimal conditions for muscle differentiation, murine G8 myoblasts expressing PPARy and C/EBPa show markedly reduced levels of the myogenic basic helix-loop-helix proteins MyoD, myogenin, MRF4, and myf5 and are completely unable to differentiate into myotubes. Under conditions permissive for adipogenesis including a PPAR activator, these cells differentiate into mature adipocytes that express molecular markers characteristic of this lineage. Our results demonstrate that a developmental switch between these two related but highly specialized cell types can be controlled by the expression ofkey adipogenic transcription factors. These factors have an ability to inhibit myogenesis that is temporally and functionally separate from their ability to stimulate adipogenesis.

Phosphoenolpyruvate carboxykinase (PEPCK) is expressed at high levels in liver, kidney, and adipose tissue. This enzyme catalyzes the rate-limiting step in hepatic and renal gluconeogenesis and adipose glyceroneogenesis. The regulatory factors important for adipose expression of the PEPCK gene are not well defined. Previous studies with transgenic mice established that the region between bp ؊2086 and ؊888 is required for expression in adipose tissue but not for expression in liver or kidney tissue. We show here that a DNA fragment containing this region can function as an enhancer and direct differentiation-dependent expression of a chloramphenicol acetyltransferase gene from a heterologous promoter in cultured 3T3-F442A preadipocytes and adipocytes. We further demonstrate that the adipocyte-specific transcription factor PPAR␥2, previously identified as a regulator of the adipocyte P2 enhancer, binds in a heterodimeric complex with RXR␣ to the PEPCK 5-flanking region at two sites, termed PCK1 (bp ؊451 to ؊439) and PCK2 (bp ؊999 to ؊987). Forced expression of PPAR␥2 and RXR␣ activates the PEPCK enhancer in non-adipose cells. This activation is potentiated by peroxisome proliferators and fatty acids but not by 9-cis retinoic acid. Mutation of the PPAR␥2 binding site (PCK2) abolishes both the activity of the enhancer in adipocytes and its ability to be activated by PPAR␥2 and RXR␣. These results establish a role for PPAR␥2 in the adipose expression of the PEPCK gene and suggest that this factor functions as a coordinate regulator of multiple adipocyte-specific genes.Phosphoenolpyruvate carboxykinase (PEPCK [EC 4.1.1.32]) catalyzes the conversion of oxaloacetate to phosphoenolpyruvate, the rate-limiting step in gluconeogenesis and glyceroneogenesis. The PEPCK gene is expressed at high levels in liver, kidney, and white and brown fat (37). Expression is regulated at the transcriptional level by multiple hormones and second messengers, including insulin, glucocorticoids, retinoic acid, thyroid hormone, and cyclic AMP (10, 21, 33). Hormonal and dietary regulation of PEPCK gene transcription is tissue specific. For example, PEPCK expression in the liver is linked to blood glucose concentration, whereas in the kidney it is primarily regulated by physiologic acid-base status. In the liver and kidney, expression is stimulated by glucocorticoids and cyclic AMP and inhibited by insulin (33). In adipose tissue, however, glucocorticoids are inhibitory (25).The complexity of its transcriptional regulation makes PEPCK an attractive model for the study of hormone-linked and tissue-specific gene expression. The regions of the PEPCK promoter important for the hormonal and dietary regulation of this gene in liver have been analyzed in detail. A complex hormone response region extending from bp Ϫ460 to Ϫ349 from the transcriptional start site contains elements important for response to glucocorticoids, insulin and retinoic acid (14,16,24,26). A thyroid hormone response element (10) and a cyclic AMP response element (31) are located a...

No abstract

Histone deacetylases (HDACs) represent an expanding family of protein modifying-enzymes that play important roles in cell proliferation, chromosome remodeling, and gene transcription. We have previously shown that recombinant human HDAC8 can be expressed in bacteria and retain its catalytic activity. To further explore the catalytic activity of HDACs, we expressed two additional human class I HDACs, HDAC1 and HDAC3, in baculovirus. Recombinant HDAC1 and HDAC3 fusion proteins remained soluble and catalytically active and were purified to near homogeneity. Interestingly, trichostatin (TSA) was found to be a potent inhibitor for all three HDACs (IC 50 value of ϳ0.1-0.3 M), whereas another HDAC inhibitor MS-27-275 (N-(2-aminophenyl)-4-[N-(pyridin-3-methyloxycarbonyl)-aminomethyl]benzamide) preferentially inhibited HDAC1 (IC 50 value of ϳ0.3 M) versus HDAC3 (IC 50 value of ϳ8 M) and had no inhibitory activity toward HDAC8 (IC 50 value Ͼ100 M). MS-27-275 as well as TSA increased histone H4 acetylation, induced apoptosis in the human colon cancer cell line SW620, and activated the simian virus 40 early promoter. HDAC1 protein was more abundantly expressed in SW620 cells compared with that of HDAC3 and HDAC8. Using purified recombinant HDAC proteins, we identified several novel HDAC inhibitors that preferentially inhibit HDAC1 or HDAC8. These inhibitors displayed distinct properties in inducing histone acetylation and reporter gene expression. These results suggest selective HDAC inhibitors could be identified using recombinantly expressed HDACs and that HDAC1 may be a promising therapeutic target for designing HDAC inhibitors for proliferative diseases such as cancer.